Unearthing Optimal Symbiotic Rhizobia Partners from the Main Production Area of Phaseolus vulgaris in Yunnan

Phaseolus vulgaris is a globally important legume cash crop, which can carry out symbiotic nitrogen fixation with rhizobia. The presence of suitable rhizobia in cultivating soils is crucial for legume cropping, especially in areas beyond the plant-host native range, where soils may lack efficient symbiotic partners. We analyzed the distribution patterns and traits of native rhizobia associated with P. vulgaris in soils of Yunnan, where the common bean experienced a recent expansion. A total of 608 rhizobial isolates were tracked from soils of fifteen sampling sites using two local varieties of P. vulgaris. The isolates were discriminated into 43 genotypes as defined by IGS PCR-RFLP. Multiple locus sequence analysis based on recA, atpD and rpoB of representative strains placed them into 11 rhizobial species of Rhizobium involving Rhizobium sophorae, Rhizobium acidisoli, Rhizobium ecuadorense, Rhizobium hidalgonense, Rhizobium vallis, Rhizobium sophoriradicis, Rhizobium croatiense, Rhizobium anhuiense, Rhizobium phaseoli, Rhizobium chutanense and Rhizobium etli, and five unknown Rhizobium species; Rhizobium genosp. I~V. R. phaseoli and R. anhuiense were the dominant species (28.0% and 28.8%) most widely distributed, followed by R. croatiense (14.8%). The other rhizobial species were less numerous or site-specific. Phylogenies of nodC and nifH markers, were divided into two specific symbiovars, sv. phaseoli regardless of the species affiliation and sv. viciae associated with R. vallis. Through symbiotic effect assessment, all the tested strains nodulated both P. vulgaris varieties, often resulting with a significant greenness index (91–98%). However, about half of them exhibited better plant biomass performance, at least on one common bean variety, and two isolates (CYAH-6 and BLYH-15) showed a better symbiotic efficiency score. Representative strains revealed diverse abiotic stress tolerance to NaCl, acidity, alkalinity, temperature, drought and glyphosate. One strain efficient on both varieties and exhibiting stress abiotic tolerance (BLYH-15) belonged to R. genosp. IV sv. phaseoli, a species first found as a legume symbiont.


Introduction
The common bean (Phaseolus vulgaris L.), known as green bean, is a leguminous plant of the genus Phaseolus [1,2].P. vulgaris is one of the frequently consumed vegetables, native to Mexico and Argentina.P. vulgaris was first domesticated in the Americas, including Mexico, Colombia, Ecuador and northern Peru, as well as in the Andean center from southern Peru to northern Argentina [3].P. vulgaris prefers warmth and is not tolerant to frost.It was only at the end of the 16th century that China began to introduce its cultivation.When P. vulgaris was introduced from the Americas, it was first introduced and domesticated in Guizhou, Yunnan, Sichuan and the surrounding provinces, before spreading to the northeast [4].According to reports, beans are grown in more than 90 countries worldwide, with a total planting area of 36.5 million km 2 and a total output of 31.4 million tons.This accounts for 50% of the total output of all edible beans, second only to the legume crop soybean.The planting area of P. vulgaris in China is 10 million km 2 , and the total output is 1.3 million tons [5,6].At present, P. vulgaris is widely cultivated in major producing areas such as northeast and north Xinjiang, and southwest China.Shandong Province and Hebei Province in north China, Heilongjiang Province and Jilin Province in northeast China and Yunnan, Guizhou and Sichuan provinces in southwest China have very rich varieties and types of beans.Yunnan is the province with the largest total planting area and output of edible beans in China, serving as the production base for high-quality fresh bean raw materials in the country.P. vulgaris has emerged as an important local cash crop because of its easy cultivation and low planting cost, resulting in a relatively extensive planting area.In some dam areas of Chuxiong, Dali, Baoshan and other places within Yunnan Province, the fresh bean industry is thriving.Cultivation of soft pod vine fresh beans has yielded substantial economic benefits, providing a new way for farmers in alpine mountainous areas to alleviate poverty and achieve prosperity.P. vulgaris is not only a vegetable, but also a grain or cash crop for foreign exchange export.Therefore, the vigorous development of bean production holds great significance for China's agriculture.
Nitrogen-fixing bacteria-known as rhizobia-are a group of Gram-negative bacteria characterized by their ability to fix nitrogen in symbiosis with legumes [7,8].When rhizobia infect leguminous plants, they induce the formation of root nodules, and within root nodules, rhizobia ultimately differentiate in bacteroids that coexist with plant cells.Bacteroids draw nutrients from plants and obtain an environment suitable for nitrogen fixation, while plants obtain nitrogen nutrients through nitrogen fixation by bacteroids, which reduce atmospheric nitrogen to ammonia forms that are assimilable by the plants [9][10][11][12][13].
There have been several studies on the diversity of rhizobia associated with P. vulgaris in China, revealing various rhizobia populations and distribution.For instance, Rhizobium vallis strains were isolated from Yunnan leguminous plants in China [31] while Rhizobium chutanense strains were isolated from P. vulgaris in Jiangxi Province [32].In addition, P. vulgaris-nodulating rhizobia resources from Heilongjiang, Liaoning and Jiangxi provinces in China were collected, and most of the isolated strains belonged to the genus Rhizobium.Among these, the species R. leguminosarum, Rhizobium laguerreae, R. phaseoli and R. vallis were identified, along with members of the genus Bradyrhizobium [33].Furthermore, the genera Sinorhizobium, Rhizobium, Bradyrhizobium, Ochrobactrum and Agrobacterium were isolated from P. vulgaris in Shaanxi Province.These included species such as R. phaseoli, R. vallis, R. giardinii, Rhizobium yanglingense, R. leguminosarum, Sinorhizobium adhaerens, S. fredii, Sinorhizobium kunmerowiae, Bradyrhizobium liaoningense, Ochrobactrum anthropic and Agrobacterium radiobacter, totaling 11 species.These findings highlight the very rich diversity of rhizobia associated with P. vulgaris [34].
Across different other countries and continents, P. vulgaris can also coexist with a wide variety of rhizobia belonging to different genotypes, genera (Rhizobium, Sinorhizobium and Bradyrhizobium) and species with dominant strains or taxa [35].In its center of origin, the dominant species among P. vulgaris nodules is R. etli.However, in some regions of Latin America, the prevalence of native strains (R. leguminosarum, R. gallicum and R. giardinii) can hinder the effectiveness of inoculation with R. etli, which is generally used to promote nodulation and nitrogen fixation.In areas where pulses have been cultivated, legume nodules harbor many species other than R. etli [24].R. tropici, characterized by high degree of genetic stability, dominates in acidic soils and regions with high-temperatures [36].In Africa, R. phaseoli, R. etli and a new Rhizobium taxon had a great advantage in forming symbiotic relationships with P. vulgaris.Meanwhile, R. leguminosarum sv.phaseoli, isolated from Moroccan soil, was found more tolerant to acidic conditions in culture media or sterile soil [18].
With the sustainable development of agriculture in China, the frequent use of chemical fertilizers and pesticides has led to environmental contamination and declining soil fertility.Nitrogen is one of the most restricted nutrients for plant growth, and nitrogen fertilizers incur significant costs in crop production [37,38].In agriculture, the symbiotic relationship between leguminous plants and rhizobia is the key to agricultural and environmental sustainability [39,40].This relationship provides a natural and renewable nitrogen resource for crops, which is economical and environmentally friendly [41,42].The rhizobia-leguminous symbiosis system, renowned for its strongest nitrogen fixation capacity, plays an important role in promoting the ecological restoration of contaminated land, including areas affected by saline conditions, heavy metals and pesticide pollution [43].Yu and Liu [44] found that enhancing soybean salt tolerance could lead to improved soybean yield in saline soils, thus highlighting the potential for biological improvement.Han [45] collected germplasm resources of elite rhizobia from broad beans in the Qinghai area, and finally screened out the strains with high drought and salt-alkali tolerance, offering a good application prospect.Chi et al. [46] conducted NaCl and drought tolerance tests on 58 peanut-nodulating rhizobia isolated from different regions of Shandong Province, and found diversity in salt and drought tolerance among rhizobia not only throughout the province but also within a same region.Wu et al. [47] screened out excellent rhizobia from soybean nodules with strong resistance to high temperatures, salt, antibiotic, acidic and alkaline conditions, as well as strong resistance to dyes and chemical drugs.Cheng et al. [48] simulated cultivated land with a history of glyphosate use by spraying different concentrations of glyphosate solution on the soil before sowing alfalfa.The results showed that different concentrations of glyphosate had inhibitory effects on the growth and nitrogen fixation of alfalfa, with the inhibitory effect strengthening with increasing glyphosate concentration.Therefore, improving crop salt tolerance, acidity and alkalinity tolerance, high temperature tolerance, drought tolerance and other traits, as well as the comprehensive development of biological treatment of difficult and constrained soils, are major issues for future agricultural development.In this study, an optimal matching experiment between rhizobia and P. vulgaris was carried out to screen out rhizobia with high efficiency in nitrogen fixation and strong abiotic stress tolerance.The aim was to improve the yield of P. vulgaris, mitigate environmental contamination caused by chemical fertilizers and pesticides and improve the quality of P. vulgaris [49,50].
Considering all the aforementioned aspects, and the fact that common bean-nodulating rhizobia in Yunnan (China) have not been systematically studied, we conducted the present study.The aim of this work was to evaluate the diversity, relative abundance and geographic distribution of native rhizobia that nodulate P. vulgaris in Yunnan Province.Traditionally, common beans have been grown largely in Yunnan as an economic crop for fresh vegetables and grains, and the cultivation area has rapidly expanded to meet consumer demand.Thus, the taxonomic status of the isolated strains from root nodules was determined through ribosomal intergenic typing, phylogenetic analyses of housekeeping genes (recA, atpD and rpoB), the 16S rRNA gene and symbiotic marker genes (nodC and nifH).Additionally, the distribution of rhizobia in relation to soil properties and environmental factors, as well as the potential of representative strains to induce effective symbiosis and tolerate abiotic stress, were investigated.

Physicochemical Characteristics of Soils and the Environment
All 15 sites differed in pH, as well as in their levels of organic matter (OM), alkaline hydrolyzable nitrogen (AN), available phosphorus (AP), available potassium and total salts (EC) (Supplementary Table S1).Soil samples ranged from acidic (five sites) to neutral (five sites) to slightly alkaline (five sites).Most of them contained an average OM level (2-4% across 11 sites), while two had a slightly low OM level (1.7-1.9%) and one a high level (>5%).The field soil at DLEY contained the highest contents of OM (62.6 g/kg soil) and AN (388.9 mg/kg soil).BSSD contained the highest content of AP (388.9 mg/kg soil) while soil from site CXMD contained the highest AK (358.3 mg/kg soil).At the opposite end, site BSCN exhibited the lowest proportions of AP and OM in soil.The field soil at CXDY had the highest salinity (EC = 819.3µS/kg soil) and site DLMD had the lowest (69.1).The sites belong to the subtropical highland climate (Cwb, Kôppen classification) with mild temperatures and dry winters.Precipitations varied from moderate (nine sites with 870-985 mm/year) to high (six sites with 1020-1350 mm/year).The altitude of the sampling sites was relatively high (1033-2967 m).

Identification of Species by Phylogenetic Analysis of Core Genes
Nearly full-length 16S rRNA genes were successfully amplified and sequenced for 46 rhizobial isolates representing all 43 IGS types and sites of origin (Table 1).The representative isolates divided into three groups (Groups 1-3) in the phylogenetic tree (Supplementary Figure S1).First of all, fifteen representative isolates clustered together with several defined Rhizobium species in Group 1 that showed 99.7-100% similarity in their 16S rRNA gene sequences.This clade comprises the type strains of Rhizobium acidisoli FH13 T , R. anhuiense CCBAU 23252 T , Rhizobium hidalgonense FH14 T and Rhizobium sophorae CCBAU 03386 T .Secondly, five representative isolates clustered in Group 2, which also included the type strains of Rhizobium dioscoreae S-93 T , and Rhizobium vallis CCBAU 65647 T .They shared 98.1-100% similarities with each other.Finally, Group 3 contained 26 representative isolates which shared 99.6-100% similarities with Rhizobium phaseoli ATCC 14482 T , Rhizobium ecuadorense CNPSo 671 T , Rhizobium chutanense C5 T , Rhizobium bangladeshense BLR175 T , Rhizobium aethiopicum HBR26 T , Rhizobium etli CFN 42 T , Rhizobium sophoriradicis CCBAU 03470 T and Rhizobium croatiense 13T T .Thus, all the representative isolates had been identified as belonging to Rhizobium.

Identification of Species by Phylogenetic Analysis of Core Genes
Nearly full-length 16S rRNA genes were successfully amplified and sequenced for 46 rhizobial isolates representing all 43 IGS types and sites of origin (Table 1).The rep-  The representative isolates were divided into thirteen clades (C1-C16) in the phylogenetic tree based on their concatenated recA-atpD-rpoB sequences (Figure 1).C1 includes nine isolates from IGS type 20 with the type strain of R. sophorae, sharing 99.1% similarities (Table 1).Thus, cluster C1 was identified as R. sophorae.Cluster C2, identified as R. acidisoli, contained R. acidisoli FH13 T and IGS type 18 representing nine isolates with 98.6% similarities.C3, identified as R. ecuadorense, contained 11 isolates of IGS type 13, showing 99% similarities with R. ecuadorense CNPSo 671 T .C5 was identified as R. hidalgonense and comprised the type strain of R. hidalgonense with IGS types 8, 25, 32, 37, 41, 42 and 43 (representing together 32 isolates (5.3% in total) sharing 97.7-100% similarities with each other).Cluster C6, identified as R. vallis, contained 14 isolates from IGS types 12 and 39, showing 98.9-99.3%similarities.C7 comprised only the IGS type 28, covering five isolates, and was identified as belonging to the species R. sophoriradicis with 98.5% similarity to type strain CCBAU 03470 T  ) contained six representative strains, and the similarity with the sequences of all known population strains was less than 97%, the model strain with the highest similarity in C4, C12 and C15 was R. chutanense, and the similarity was only 94.6-96.3%, and the model bacteria with the highest similarity between C10 and C16 was R. phaseoli, with a similarity of only 92.9-96.3%, is suspected to be a new population of rhizobia strains of such groups C4, 10, 12, 15 and 16.The phylogenetic analyses of the single gene of recA, atpD or rpoB are shown in Supplementary Figures S2-S4.

Identification of Symbiovars by Phylogenetic Analysis of Symbiotic Genes
Besides the core genes studied above to taxonomically identify rhizobia, it is complementary to type rhizobia according to their symbiovar related to the legume-host spectrum, which does not follow taxonomic phylogeny.To do so, common genes in-

Identification of Symbiovars by Phylogenetic Analysis of Symbiotic Genes
Besides the core genes studied above to taxonomically identify rhizobia, it is complementary to type rhizobia according to their symbiovar related to the legume-host spectrum, which does not follow taxonomic phylogeny.To do so, common genes involved in nodulation (nodC coding for an N-acetyltransferase) and fixation (nifH coding for a subunit of nitrogenase) were investigated in the 46 representative strains.

Correlation Analysis of Rhizobial Distribution with Soil and Environmental Properties
PCA was used to explore the relationships between soil and environment properties, and the rhizobial community composition based on IGS genotypes.The PCA results (Figure 4) showed that the soil chemical factors had different effects on the distribution of the rhizobia populations and IGS types.The IGS types 3, 6, 15, 17, 19, 20, 23, 30 and 43 (left upper part of Figure 4) included 114 isolates recovered mainly in CXDY, CXYA, BSSD, BSCN and BSLY (Supplementary Table S2).Their distribution was positively correlated with pH, EC and average rainfall (AvePrecp), and negatively related with soil OM contents.In particular, IGS20 and IGS15 showed a positive correlation with pH and EC, while IGS3 and IGS20 were associated with the average rainfall (Figure 4).The IGS types 1, 7, 11, 22, 25, 27, 29, 32, 34, 37 and 38 (lower left part of Figure 4) gathered the majority of isolates (222) distributed across four main sites (CXLF, DLEY, DLMD and DLWS) (Supplementary Table S2); they presented a positive association with AN, pH and OM, but were negatively correlated with AK values.In particular, the IGS27 and IGS29 were positively correlated with AN, while IGS37 and IGS7 correlated with higher OM values (Figure 4).The IGS types 5, 8, 10, 13, 24, 35, 36 and 42 (including 82 strains) (lower right part of Figure 4) tended to be associated with DLXY (Supplementary Table S2).Meanwhile, these strains were positively correlated with OM and negatively correlated with EC, pH value and average rainfall.Notably, the IGS10 was positively correlated with OM values (Figure 4).Finally, the IGS types 2, 4, 21, 12, 9, 31, 39, 18, 28, 33, 16, 14, 26, 40 and 41 (middle and upper right part of Figure 4), representing 190 isolates mainly from CXNH, CXDH, CXSB, CXMD and CXWD, were positively distributed with AK, but lower AN, EC and pH values.In particular, IGS28 was positively correlated with AK values.Thus, we found that the different rhizobial populations were influenced differently by edaphic factors, showing potential positive or negative significant correlations.
R. sophoriradicis, R. acidisoli, R. vallis, R. etli and R. genosp.II were mainly distributed in CXWD, CXSB, CXDH, CXNH and CXMD.Their distribution was positively correlated with the available potassium content in the soil and negatively correlated with electrical conductivity and pH value.R. phaseoli, R. chutanense, R. hidalgonense, R. ecuadorense, R. genosp.I and R. genosp.III were primarily distributed in the sampling points of DLXY, DLWS, DLMD and DLEY, with a positive correlation with organic matter content and negatively correlated with average precipitation.R. sophorae and R. anhuiense were mainly distributed in CXDY, CXLF and BSSD, with a positive correlation with alkaline nitrogen, electrical conductivity, and pH value, and a negative correlation with available potassium content.R. croatiense, R. genosp.IV and R. genosp.V were primarily distributed in CXYA, BSCN and BSLY.Their distribution was positively correlated with average precipitation and negatively correlated with the content of organic matter and alkaline nitrogen in the soil.Furthermore, R. anhuiense was widely distributed in all 13 sampling points, indicating that these strains were with strong adaptability to different conditions(Supplementary Figure S10).R. sophoriradicis, R. acidisoli, R. vallis, R. etli and R. genosp.II were mainly distribute in CXWD, CXSB, CXDH, CXNH and CXMD.Their distribution was positively correlate with the available potassium content in the soil and negatively correlated with electric conductivity and pH value.R. phaseoli, R. chutanense, R. hidalgonense, R. ecuadorense, genosp.I and R. genosp.III were primarily distributed in the sampling points of DLX DLWS, DLMD and DLEY, with a positive correlation with organic matter content an negatively correlated with average precipitation.R. sophorae and R. anhuiense were main distributed in CXDY, CXLF and BSSD, with a positive correlation with alkaline nitroge electrical conductivity, and pH value, and a negative correlation with available pota sium content.R. croatiense, R. genosp.IV and R. genosp.V were primarily distributed CXYA, BSCN and BSLY.Their distribution was positively correlated with average pr cipitation and negatively correlated with the content of organic matter and alkaline n trogen in the soil.Furthermore, R. anhuiense was widely distributed in all 13 samplin
The determination of abiotic stress tolerance across 46 symbiotic strains of P. vulgaris showed a rich diversity in terms of adaptability among the different strains.The comprehensive experimental results, encompassing all the abiotic stress tested, i.e., acidity, alkalinity, NaCl, temperature, PEG and glyphosate tolerance, revealed CWDB-3, DXYH-4, BLYH-17 and BLYB-15 as the most tolerant rhizobial strains (Supplementary Table S8).Combined with symbiotic assay results (Section 2.6), BLYB-15 (belonging to R. dioscoreae, IGS type 17 according to Section 2.3.)emerged as the strain with both high potential symbiotic efficiency scores on v1 and v2 local bean varieties, together with notable environmental stress tolerance potential (growth at pH 6, pH 11, 45 • C, with 1% NaCl, 10% PEG or 1.8 mL/L glyphosate).

Discussion
The study of P. vulgaris nodulating rhizobia in Yunnan, combined with the examination of soil physicochemical properties and associated environmental factors, revealed the biogeographic distribution of rhizobia in the area.Unlike previous studies, we systematically investigated the diversity of P. vulgaris symbionts at 15 sampling sites in Chuxiong, Dali, and Baoshan areas of Yunnan Province.A total of 608 rhizobial isolates were thus obtained from root nodules and characterized genetically and symbiotically.Taking all the results from the IGS PCR-RFLP typing, phylogenies of 16S rRNA gene sequences, concatenated recA-atpD-rpoB sequences, as well as nodC and nifH sequences, the isolates obtained in this study were classified as a diverse community consisting of 43 IGS types within 16 species all within the genus Rhizobium.Most of the representatives selected by IGS type and site belonged to the sv.phaseoli and a minority to sv. viciae.The dominant species among common bean-nodulating rhizobia were R. anhuiense (28.8% in relative abundance) and R. phaseoli (28.0%), followed by R. croatiense (14.8%) and R. hidalgonense (5.3%).These species were found to be widely distributed across most of the fifteen sites (11-13 sites).The twelve other species (R. sophorae, R. acidisoli R. ecuadorense, R. vallis, R. sophoriradicis, R. etli, R. chutanense and Rhizobium genosp.I~V) were less represented with a narrower distribution (<5.6% and restricted to 1-7 sites).
The data provide new insights on the diversity structure and geographic distribution of P. vulgaris-symbiotic rhizobia in China and areas outside of the plant-host native range.Among the recovered species in Yunnan areas, R. sophorae was initially isolated from effective nodules of the shrubby Sophora (Sophora flavescens) in Changzhi City (Shanxi Province, China), and was found to be able to effectively nodulate not only Sophora but also P. vulgaris [51].This rhizobial species was later recovered from root nodules of Vicia faba L. grown in Panxi (southwest China) [52], southwest China [53], and Hebei Province (northeast China) [54,55].These findings underscore the widespread existence of R. sophorae, emphasizing its adaptability to diverse soil and environmental conditions in China.R. acidisoli, initially isolated from P. vulgaris nodules in acidic soils of Mexico and forming symbiosis with P. vulgaris [15], was later discovered in Morocco [56].R. ecuadorense, isolated from P. vulgaris in northern and central Ecuador, had a valuable effective nitrogen fixation with P. vulgaris [21].R. hidalgonense was isolated from P. vulgaris nodules in the State of Mexico [20].R. anhuiense was first described in rhizobia of pea and V. faba in Anhui and Jiangxi Provinces [57], and has been widely recorded in several other provinces in China (provinces of Shandong [58], Sichuan [59] and Hebei [55]).The present study, with a total of 28% relative abundance detected over 13 Yunnan sites, suggests a wide geographic distribution of R. anhuiense in China.R. croatiense was isolated from P. vulgaris landraces from soils of northeast Croatia [16].R. vallis, isolated from nodules of three legume plants (P.vulgaris, Mimosa mimosa and Indigofera spicata) grown in the Yunnan province of China, effectively nodulated P. vulgaris but did not nodulate M. mimosa and I. spicata, suggesting that R. vallis could be endophytic in some root nodules [31].R. sophoriradicis, originally isolated from Sophora (S. flavescens) [51], has been extensively studied in P. vulgaris in Iran [60], South Africa [61] and Peru [62], indicating its widespread distribution among plant species.R. phaseoli, which predominates in native areas of common beans, was isolated in Mexico [63], but also from non-native countries such as Ethiopia [26], Brazil [64] and Eswatini [65].R. chutanense was isolated from P. vulgaris in Jiangxi Province for the first time, and can effectively trigger nodulation with both P. vulgaris and soybean [32].R. etli, rarely found in this study (<1%), was initially discovered in root nodules of the Mexican leguminous plant Mimosa affinis and was able to form nodules and fix nitrogen on M. mimosa [66].This species was subsequently found in P. vulgaris nodules collected across different agro-ecological zones in Senegal, Gambia (West Africa) [67] and northwestern Argentina.It emerged as a predominant species in common bean nodules from P. vulgaris origin areas [24,68] and from different regions of Jordan [69] and Brazil [70].Moreover, R. etli was widely found to be tolerant to high salinity and pH levels in northwestern Morocco [71], Ethiopia [26,72] and Egypt [73,74].These results show the wide distribution of rhizobia in time and space, as well as its high adaptability to different soil and environmental factors.Adding to the diversity distribution.Notably, R. tropici was not observed in this study, similar to findings in the Shaanxi Province of China [34].However, R. tropici, along with R. etli and R. phaseoli, is another predominant species among P. vulgaris symbionts widely recovered in continents and countries such as Columbia [14], Argentina [68], Brazil [64,75] and Iran [60], as well as in north, west or South Africa [61,67,75].Bacteria of the genus Rhizobium have been isolated from a variety of sources and widely utilized for their nitrogenfixing abilities, both in environment and agriculture.Our findings highlight the broad geographic distribution of these species worldwide, suggesting that P. vulgaris plants have selected chromosomal backgrounds or communities suited to local conditions over longterm legume cultivation.Ultimately, the detection of highly conserved nodC genes across the 13 rhizobial species identified in this study provides further evidence of P. vulgaris plants' stringent selection of symbiosis genes in their microsymbionts.This selective pressure may favor symbiosis genes toward the most adapted indigenous rhizobia, as reported in other cases [76].To sum up, our results underscore the necessity of screening and selecting high-quality rhizobial strains with strong adaptability to local conditions for inoculant production aimed at enhancing P. vulgaris and other legume yields.
In conclusion, our study demonstrated the existence of indigenous rhizobia that form an effective symbiosis with P. vulgaris cultivated in southwest China, in which R. anhuiense was newly recorded as P. vulgaris-nodulating rhizobia.R. phaseoli, R. anhuiense, R. croatiense and R. hidalgonense were the most abundant and widely distributed in the studied soils (28.0%, 28.8%, 14.8% and 5.6%) and formed unique species assemblage of P. vulgaris-nodulating rhizobia along with twelve less frequent species (R. sophorae, R. acidisoli, R. ecuadorense, R. vallis, R. sophoriradicis, R. etli, R. chutanense and Rhizobium genosp.I~V).Moreover R. anhuiense occurred in thirteen tested soil types, indicating that strains of these species could be better competitors and adapted to different soil conditions.Finally, all strains tested belonged to the sv.phaseoli regardless of their species affiliation or sv.viciae associated with R. vallis.Through symbiotic experiments, the number of nodules per plant was good with chlorophyll index higher than controls for most strains.Dry weights of host plants were significantly improved under inoculation for about half of the P. vulgaris-nodulating strains.Therefore, inoculation with rhizobia associated with P. vulgaris can significantly promote the growth of P. vulgaris plants and achieve effective symbiosis.In parallel, representative strains to IGS types and sites were screened for abiotic stress tolerance, aiming to potentially assist the host legume to cope with soil and environmental stresses.A total of four strains with strong comprehensive tolerance were identified.Combined with the results of symbiotic assay on both P. vulgaris varieties, the strain BLYB-15 (R. genosp.IV sv.phaseoli, IGS type 17) was found to exhibit high symbiotic efficiency and strong stress tolerance.The resource collection of common bean-associated rhizobia in Yunnan Province, analyzed for genetic and phenotypic diversity, provides significant guidelines for increasing local and sustainable P. vulgaris production, with the potential for site-specific selection of efficient rhizobial genetic types.

Field Soil Sampling and Soil and Environmental Characteristics
Soils were sampled from fields cultivated with local common beans (Phaseolus vulgaris L.) located in the cities of Chuxiong (CX), Dali (DL) and Baoshan (BS), all in the province of Yunnan, southwest China.A total of 15 sites were studied: 8 at CX (CX-MD, YA, DY LF, DH, WD, SB, NH), 4 at DL (DL-XY, WS, MD, EY) and 3 at BS (BS-LY, CN, SD) (Supplementary Table S1).At each site, soil was collected from the main production area of P. vulgaris to a depth of 10-20 cm near the P. vulgaris roots during its flowering stage in June 2022.For each site, three randomly taken soil sub-samples of equal volume were crushed to a uniform state, and transported to the laboratory in an ice-filled cooler [77].Part of each soil sample was chemically analyzed for pH, electrical conductivity (EC), organic matter (OM), available phosphorus (AP), available potassium (AK) and alkaline hydrolyzable nitrogen (AN), as described previously [78].Using the location information of the sampled sites, annual climate data were collected for each site using DIVA-GIS software Version 7.5 (University of California, Los Angeles, CA, USA).This includes the altitude of the sampling site (Alt), the amount of rainfall throughout the year (AvePrecp), the maximum (AveTmax) and minimum temperatures (AveTmin) and the analysis of climate data [79].

Rhizobial Isolation and Conservation
Surface-sterilized seeds (2.5% w/v NaClO solution for 5 min) of the common bean (variety 1 of black P. vulgaris: garden bean and variety 2 of white P. vulgaris: edible pod bean from the local areas of Yunnan Province) were germinated and the seedings were sown in surface-sterilized plastic pots (15 cm high × 10 cm diameter) filled with each sampled soil mixed with sterilized vermiculite (1/5 v/v).The three randomly taken soil sub-samples were thoroughly mixed to constitute a representative soil sample per field site.For each representative soil sample, 10 repetitions of tracking experiments were performed.All plants were grown under greenhouse conditions of 25/20 • C (day/night) with a 16 h photoperiod.Sterilized water was added to the pots throughout the experiment as required.After 45 days, all plants in all soils were uprooted and bacterial strains were isolated from nodules according to the standard protocol [53].Five plants from each sampled soil were selected for excision of root nodules and isolation of rhizobia.Root nodules were surfacesterilized, then each individual sterilized root nodule was crushed in sterile water and the bacterial suspension was streaked onto a Yeast extract Mannitol Agar (YMA) plate.After incubation at 28 • C for 2 to 3 days, single colonies representing the dominant bacteria on each plate were picked up and purified by cross-streaking on new YMA plates until pure cultures were visually obtained.All purified isolates were conserved in Tryptone Yeast (TY) broth (tryptone 5 g; yeast extract 3 g; CaCl 2 0.6 g; distilled water 1 L, pH 7.0) supplied with glycerol (20%, v/v) and stored at −80 • C for long-term storage.Additionally, they were maintained on YMA slants at 4 • C for temporary storage.

Genomic Characterization of Rhizobial Isolates
The genomic DNA of each isolate was purified according to Terefework et al. [80,81].DNA was used as template for PCR amplifications of the 16S-23S rRNA intergenic spacer (IGS) region with primers IGS1490 (forward, TGCGGCTGGATCACCTCCTT) and IGS132 ′ (reverse, CCGGGTTTCCCCATTCGG) [82].PCR amplification was carried out in a standard 50 µL reaction mixture, including 1 µL of DNA template and 5 U of Taq DNA polymerase (Sangon Biotech (Shanghai) Co., Ltd., Shanghai, China).Aliquots of amplified PCR products (900 base pairs) were visualized after electrophoresis in a 1.0% (w/v) agarose gel labeled with GoldView type I.Then, PCR products were digested separately with the endonucleases HaeIII, MspI and HhaI [83] at 37 • C for 10 h.The 16S-23S rRNA gene IGS type of each strain was designated after separation and visualization of restriction fragments by electrophoresis in 2.5% (w/v) agarose gel and UV-illumination.

Molecular and Phylogenetic Identification of the Isolates, Alpha-Diversity Estimation
Isolates sharing the same RFLP pattern of 16S-23S rRNA gene IGS in this study were designed as an IGS type.One representative strain for each IGS type in each sample site (total of 46 strains) was selected for amplification of the 16S rRNA gene using the forward primer P1 (CGGGATCCAGAGTTTGATCCTGGTCAGAACGCT) and reverse primer P6 (CGGGATCCTACGGCTACCTTGTTAC GACTTCACCCC3) [82].The PCR products were verified as mentioned above, and were sent for commercial sequencing based on the Sanger method (Sangon Biotech (Shanghai, China) Co., Ltd.).The acquired sequences were compared against the NCBI database using the online BLASTN tool, and sequences for type strains of defined Rhizobium species sharing similarities greater than 97.0% with the new isolates were extracted.The phylogenetic analysis was conducted in the MEGA 7.0 software [84].Sequences were aligned using Clustal W and the best model of sequence evolution was selected.Then, the phylogenetic tree was inferred using the maximum likelihood (ML) and the non-parametric bootstrap (500 pseudo-replications) methods.
The sequences have been deposited in the NCBI database (accession numbers are indicated on the trees (Figures 1 and 2 and Supplementary Figures S1-S5)).
Alpha-diversity was calculated to estimate the effective numbers of species (ENS) using three diversity indices (numbers of clusters, exponential Shannon index and the inverse of the Simpson index [90]).

Correlation Analysis of Soil Properties and Environmental Factors with Rhizobial Communities
The principal component analysis (PCA) was performed using CANOCO version 5.0 [91] to investigate the relationships between soil properties (AN, AP, AK, OM, EC and pH) and environmental factors (Alt, AveTmin, AveTmax and AvePrecp) and the rhizobial community composition based on IGS genotypes and rhizobial species.The distance matrix generated from the response variable (i.e., rhizobial composition data) were based on percentage dissimilarity (i.e., Bray-Curtis dissimilarity) obtained from the 43 IGS genotypes (used to identify rhizobia).This matrix was then correlated to environmental and soil factors.

Symbiotic Efficiency Measurements
Symbiotic efficiency of the representative strains was evaluated on both variety 1 and variety 2 of the local common bean.Briefly, surface-sterilized seedlings were aseptically transferred in pots (1 plant/pot) containing sterile vermiculite as substrate and inoculated with 1 mL of rhizobial suspension (OD 600 = 1.0).Plants were grown under greenhouse conditions and watered with sterile N-free nutrient solution as required [92].Symbiotic performance was evaluated 45 days after inoculation for both bean varieties.Common bean growth was estimating by weighting their dry root and shoot biomass, counting the number of nodules and measuring leaf chlorophyll contents (SPAD chlorophyll meter).Uninoculated plants were included as a negative control, and all treatments were performed in three replicates.A symbiotic performance score (%) was calculated for each treatment, considering 25% of the nodulation index (number of nodules per plant), 25% of the leaf chlorophyll index (SPAD value per plant) and 50% of the plant biomass index (dry roots and leaves per plant).Each performance index was standardized by dividing it by the best average value obtained in the experiment (n = 3).Data were analyzed by one-way ANOVA followed by an LSD post hoc test (p = 0.001).

Measurements of Abiotic Stress on Rhizobial Strain Growth
Acid and alkali tolerance test: The 46 representative strains of common bean-nodulating rhizobia were inoculated on YMA solid medium at different pH (5, 6, 7, 8, 9, 10 and 11).Each strain was repeated on 3 plates and incubated at 28 • C. Growth results were recorded after 2 and 3 days in comparison with the control at pH 7.
Salt tolerance test: Representative rhizobial strains were inoculated on YMA solid medium containing different concentrations of NaCl (0.01%, 1%, 2%, 3% and 4% (w/v)), with 0.01% NaCl plates as control.Each strain was tested in triplicate, and growth was assessed after 2 and 3 days of incubation at 28 • C.
Temperature growth tolerance test: Each representative rhizobium was inoculated in liquid TY medium and cultured at different temperatures (4 • C, 10 • C, 28 • C, 37 • C and 45 • C).Each treatment was replicated 3 times.Optical density (O.D.) was measured at 600 nm after 2 and 3 days of incubation.
Drought tolerance test: Polyethylene glycol 6000 (PEG 6000) was used to artificially simulate drought conditions.Different amounts of PEG were added to YM broth medium, resulting in final concentration of 0%, 3%, 5%, 7%, 10% and 15% (w/v).Each rhizobium was inoculated into these media (three replicates), and cultures were incubated in a rotary incubator at 28 • C for 2 and 3 days.
Glyphosate tolerance test: Glyphosate was sourced from a commercial product containing 41% (w/v) glyphosate isopropylamine saline solution in H 2 O, at an effective concentration of 2.16 M. Each rhizobial strain was inoculated on YMA medium supplemented

Figure 1 .
Figure 1.Maximum likelihood phylogenetic tree based on concatenated recA-atpD-rpoB gene sequences (1185 base pairs) showing the relationships of rhizobia isolated from Phaseolus vulgaris L. in Yunnan Province of China.The tree was constructed under the best-fit model (GTR + G + I).Scale bar indicates 0.02 nt substitution per site.Bootstrap confidence values (%) calculated for 500 replications > 50% are indicated at the internodes.

Figure 3 .
Figure 3. Maximum likelihood phylogenetic tree based on symbiotic gene nodC (376 base pairs)showing the relationships of the rhizobia isolated from nodules of Phaseolus vulgaris L. in Yunnan Province of China.The two nodC groups found among isolates are named N1 and N2.The tree was constructed using the maximum likelihood method under the best-fit model (T92 + G + I).Scale bar indicates 0.05 nt per site.Bootstrap confidence values (%) calculated for 500 replications > 70% are indicated at the internodes.

Figure 4 .
Figure 4. PCA to relate the distribution of the 42 IGS types of isolates (n > 1) to physicochemic factors of soils and environment collected from the different sites.The blue arrows indicate IG types of rhizobia, green indicate the sampling sites and red arrows represent soil properties an environmental factors.The longer the arrow was, the greater the influence of the soil property an environmental factor presents on the distribution of the IGS types.The smaller the angle betwe the arrow and the IGS type was, the stronger the effect of the soil property or environmental fact on distribution of the IGS type.

Figure 4 .
Figure 4. PCA to relate the distribution of the 42 IGS types of isolates (n > 1) to physicochemical factors of soils and environment collected from the different sites.The blue arrows indicate IGS types of rhizobia, green indicate the sampling sites and red arrows represent soil properties and environmental factors.The longer the arrow was, the greater the influence of the soil property and environmental factor presents on the distribution of the IGS types.The smaller the angle between the arrow and the IGS type was, the stronger the effect of the soil property or environmental factor on distribution of the IGS type.

Table 1 .
Genetic groupings of Rhizobium isolates associated with Phaseolus vulgaris and their geographical distribution in the different sampling sites.